Optics collection and detection system and method

ABSTRACT

Optics collection and detection systems are provided for measuring optical signals from an array of optical sources over time. Methods of using the optics collection and detection systems are also described.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.61/306,235 filed Feb. 19, 2010 and entitled INTEGRATED ANALYTICALDEVICES AND SYSTEMS, U.S. Provisional Patent Application No. 61/387,916filed Sep. 29, 2010 and entitled INTEGRATED ANALYTICAL SYSTEM ANDMETHOD, U.S. Provisional Patent Application No. 61/410,189 filed Nov. 4,2010 and entitled ILLUMINATION OF INTEGRATED ANALYTICAL SYSTEMS, andU.S. patent application Ser. No. ______ [Attorney Docket No.067191-5044-US] filed ______ and entitled OPTICS COLLECTION ANDDETECTION SYSTEM AND METHOD, the entire contents of which applicationsis incorporated herein for all purposes by this reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to optics collection and detection systems andmethods for their use.

2. Description of Related Art

A large number of systems for optical analysis of samples or materialsemploy complex optical trains that direct, focus, filter, split,separate and detect light to and/or from the sample materials. Suchsystems typically employ an assortment of different optical elements todirect, modify, and otherwise manipulate light directed to and/orreceived from a reaction site.

Conventional optical systems typically are complex and costly. Thesystems also tend to have significant space requirements. For example,typical systems employ mirrors and prisms in directing light (e.g. laserlight) from its source to a desired destination for detection.Additionally, such systems may include light splitting optics such asbeam splitting prisms to generate two beams from a single original beam.In the case of modern analysis systems, there is a continuing need forsystems with very high throughput and portability.

There is a continuing need for optical systems for collecting anddetecting excitation signals. For example, analytical systems formonitoring processes at the single molecule level show great promise butrequire the collection and detection of small levels of illumination.There is a need for collecting and detecting optical signals, andspecifically for collection and detection systems with reduced noise andimproved performance.

There is a continuing need to improve upon the functionality, footprintand cost of systems for optical analysis. The present invention providesdevices, systems, and methods for overcoming the above problems inaddition to other benefits.

BRIEF SUMMARY

One aspect of the present invention is directed to an integrated devicefor measuring optical signals from an array of optical sources overtime. The device may include an array of elements, each having a toplayer comprising an optical source that emits two or more opticalsignals, each optical signal comprising different wavelengths, a middlelayer comprising a spectral diversion element, and a bottom layercomprising a detector sensitive to spatial distributions of light. Eachspectral diversion element may divert each of the two or more opticalsignals onto different regions of the bottom layer, whereby the identityof the optical signal can be identified by the relative spatial lightintensity across the detector.

The optical source may include a zero mode waveguide and the opticalsignals may be emitted from fluorescent labels corresponding to achemical or biochemical reaction occurring within the zero modewaveguide. The chemical or biochemical reaction may include nucleic acidsynthesis. The spectral diversion element may include an optical gratingor holographic element. The spectral diversion element may include aBragg grating disposed at an angle to the normal to the central ray ofthe emitted light. The pixels may be in a linear array. The pixels maybe in a two dimensional array. The four different optical signals may beemitted from each optical source. The detector has four pixels, eachcorresponding to one of the four optical signals whereby the spectraldiversion element diverts each of the colors to a different element. Thespectral diversion element may include a lens. The lens may becylindrically symmetrical and diverts different wavelengths of light atdifferent angles from the center of the lens resulting in a circularlysymmetric pattern on the detector for each set of wavelengths. Thedetector may include one central pixel and one or more pixels comprisinga circular ring around the central pixel.

Another aspect of the present invention is directed to a pixel includinga photodiode having at least a first and a second transfer gate, whereinthe pixel may be configured such that the photodiode sends charge to onetransfer gate for a first period of time, then send charge to a secondtransfer gate for a second period of time before the remaining chargefrom the pixel is unloaded.

The pixel further may include a third transfer gate and a fourthtransfer gate wherein after the second period of time, charge may besent to the third transfer gate for a third period of time, then chargemay be sent to the fourth transfer gate for a fourth period of timebefore the charge from the pixel is unloaded.

Another aspect of the present invention is directed to a systemincluding an excitation light source that emits a first wavelength rangefor a first excitation time, then emits a second wavelength range for asecond excitation time, the excitation light source providing excitationlight to a sample, the sample having a first fluorophore that is excitedby the first wavelength range, and a second fluorophore that is excitedby the second wavelength range, and a pixel comprising a photodiodehaving at least a first transfer gate and a second transfer gate,wherein the pixel is configured such that the photodiode sends charge tothe first transfer gate for a first collection time, then send charge tothe second transfer gate for a collection time before the charge fromthe pixel is unloaded. The system may be configured such that the firstexcitation time corresponds to the first collection time and the secondexcitation time correlates to the second collection time, whereby theemission from the first fluorophore can be distinguished from theemission from the second fluorophore.

The light source may further emit a third wavelength range for a thirdexcitation time, then emit a fourth wavelength range for a fourthexcitation time, wherein the sample further may include a thirdfluorophore that is excited by the third wavelength range, and a fourthfluorophore that is excited by the fourth wavelength range, and thephotodiode further may include a third transfer gate and a fourthtransfer gate. The pixel may be configured such that the photodiodesends charge to the third transfer gate for a third collection time,then send charge to the fourth transfer gate for a collection timebefore the charge from the pixel is unloaded. The system may further beconfigured such that the third excitation time corresponds to the thirdcollection time and the fourth excitation time correlates to the fourthcollection time, whereby the emission of each of the four fluorophorescan be distinguished. The excitation light may include a first laserthat emits the first wavelength range and a second laser that emits thesecond wavelength range, a third laser that emits the third wavelengthrange, and a fourth laser that emits a fourth wavelength range.

Yet another aspect of the present invention is directed to a methodincluding illuminating a sample with a first wavelength range for afirst excitation time, then illuminating the sample with a secondwavelength range for a second excitation time, wherein the sample mayinclude a first fluorophore that is excited by the first wavelengthrange, and a second fluorophore that is excited by the second wavelengthrange, and directing emitted light from the sample to a single pixelthat measures light for a first collection time and then measures lightfor a second collection time, such that the pixel separately storescharge related to each of the collection times whereby the chargerelated to each of the collection times can be separately read out,wherein the first excitation time corresponds to the first collectiontime and the second excitation time correlates to the second collectiontime, whereby the emission from the first fluorophore can bedistinguished from the emission from the second fluorophore.

A further aspect of the present invention is directed to an integrateddevice for measuring optical signals from an array of optical sourcesover time. The device may include an array of elements, each elementhaving a top layer comprising an optical source that emits two or moreoptical signals, each optical signal having a different rate of signaldecay, a middle layer capable of transferring light from the top layerto the bottom layer, and a bottom layer comprising a detector having asingle pixel. The pixel may measure the characteristic photonic emissionlifetime of each of the two or more different optical signals, allowingthe pixel to distinguish the identity of each of the optical signals.

At least one of said optical sources may include a zero mode waveguide.At least one of said optical sources may include a chemical orbiochemical reaction, and the two or more optical signals may be fromfluorescent labels whose fluorescence is indicative of the occurrence ofthat reaction.

Still a further aspect of the present invention is directed to anintegrated device for measuring optical signals from an array of opticalsources over time, the device comprising an array of elements, eachelement including a top layer comprising an optical source that emitstwo or more optical signals, each optical signal having a different rateof signal decay, a middle layer capable of transferring light from thetop layer to the bottom layer, and a bottom layer comprising a detectorhaving a single pixel. The pixel may measure the characteristicabsorption depth of each of the two or more different optical signals,allowing the pixel to distinguish the identity of each of the opticalsignals.

The optical source may include a zero mode waveguide. The optical sourcemay include a chemical or biochemical reaction, and the two or moreoptical signals may be from fluorescent labels whose fluorescence isindicative of the occurrence of that reaction. The pixel may be astacked diode. The detector may include multiple proximal pixels. Thedetector may include a single position sensitive pixel or a positionsensitive diode.

Yet another aspect of the present invention is directed to an opticscollection and detection system including a reaction cell, anillumination light source providing illumination light to the reactioncell at a first wavelength λ1, a detector for detecting excitation lightat a second wavelength λ2, and a photon band gap (PBG) layer disposedbetween the reaction cell and the detector, wherein the PBG layerrejects light at the first wavelength λ1 but allows light at the secondwavelength λ2 travel toward the detector.

The optics collection and detection system may further include aplurality of detectors for detecting excitation light, wherein one ofsaid detectors detects light at the second wavelength λ2, and another ofsaid detectors detects light at a third wavelength λ3, a plurality ofPBG stacks, each disposed between the PBG layer and a respectivedetector, wherein one of said PBG stacks rejects light at the thirdwavelength λ3 but allows light at the second wavelength λ2 to traveltoward said one detector, and wherein another of said PBG stacks allowslight at the second wavelength λ2 but allows light at the thirdwavelength λ3 to travel toward said other detector. The system mayinclude four detectors, each formed by a sensor quadrant of a quadphotodiode (QPD), and four PBG stacks, wherein each PBG stack may beoptically aligned with a respective sensor quadrant. Each detector maybe formed by a respective region of a PIN diode. The optics collectionand detection system may further include a doped region in which a heavymetal or a semiconductor is doped into fused silica or silicon oxideadjacent the reaction cell to provide an index of refraction gradient todisperse emission light at different angles leaving the reaction cell.

Yet another aspect of the present invention is directed to a method offabricating optics collection and detection system including providing asubstrate, applying a first lithographic protection mask to form aprotective dome (PD), applying an anisotropic etch to form ahemispherical cavity, applying thin film deposition to form a filter,applying a second lithographic protection mask to protect thehemispherical cavity, applying a planarization layer, applying a toplayer, and etching the top layer to form a reaction cell above thehemispherical cavity. A hemispherical filter may be formed adjacent abottom surface of the reaction cell.

And yet another aspect of the present invention is directed to a methodof DNA sequencing including affixing a polymerase to a photodiode,labeling a nucleotide with a metal nanoparticle, exciting a plasmonresonance in the metal nanoparticles, and monitoring the level ofbaseline current in the photodiode.

The methods and apparatuses of the present invention have other featuresand advantages which will be apparent from or are set forth in moredetail in the accompanying drawings, which are incorporated herein, andthe following Detailed Description of the Invention, which togetherserve to explain certain principles of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic view of an exemplary optics collection anddetection system and method in accordance with various aspects of thepresent invention.

FIG. 2 is a perspective rendering of an exemplary optics collection anddetection device of the system of FIG. 1.

FIG. 3 is a schematic view of an exemplary optics collection anddetection device of FIG. 1.

FIG. 4A is a schematic view of quad photodiode (QPD), and FIG. 4B andFIG. 4C are schematic top and side views of another photodiode, both ofwhich are suitable for use with the device of FIG. 3.

FIG. 5 is a schematic cross-sectional representation of an exemplaryintegrated structure suitable for use with the system of FIG. 1.

FIG. 6 is a graph showing an exemplary absorption coefficient ofsilicon.

FIG. 7 is a graph showing an exemplary detector absorption curve.

FIG. 8 is schematic representation of an exemplary collimator and prismsuitable for use with the system of FIG. 1.

FIG. 9( a) to FIG. 9 m) is a schematic sequence illustrating anexemplary method of fabricating optic filter layers suitable for usewith the system of FIG. 1.

FIG. 10A and FIG. 10B are schematic representations of an exemplarypolymerase affixed to a photodiode suitable for use with the system ofFIG. 1.

FIG. 11 is a schematic representation of an array of reaction chamberssuitable for use in the system of FIGS. 1.

FIG. 12 is a schematic representation of a typical imaging pixel.

FIG. 13 is a schematic representation of an array of analytic devicessuitable for use in the system of FIG. 1.

FIG. 14A and FIG. 14B are a schematic top and side views, respectively aFaraday cage suitable for use in the system of FIG. 1.

FIG. 15( a) to (g) is a schematic sequence of illustrating an exemplarymethod of fabricating an analytic device suitable for use in the systemof FIG. 1.

FIG. 16 is a schematic representation of an exemplary analytic devicesuitable for use in the system of FIG. 1.

FIG. 17( a) to (i) are schematic sequences illustrating exemplarymethods of fabricating analytic devices suitable for use in the systemof FIG. 1.

FIG. 18( a) to (d) is a schematic sequence illustrating an exemplarymethod of fabricating an analytic device, along with a plan viewthereof, suitable for use in the system of FIG. 1.

FIG. 19( a) to (d) is a schematic sequence illustrating an exemplarymethod of fabricating an analytic device, along with a plan viewthereof, suitable for use in the system of FIG. 1.

FIG. 20( a) to (c) are schematic representations illustrating anexemplary analytic device suitable for use in the system of FIG. 1.

FIG. 21 is a schematic representation of an active illuminated systemsuitable for use in the system of FIG. 1.

FIG. 22 is a schematic representation of an imaging system withselectable illumination wavelengths suitable for use in the system ofFIGS. 1.

FIG. 23 is a schematic representation of an integrated photodiodesuitable for use in the system of FIG. 1.

FIG. 24 is a graph illustrating an exemplary time series of a sampledevent with 0.6e/sample and 1e-background.

FIG. 25 is a graph illustrating an exemplary result of TDM Pulseextraction with signal=0.5 electron/sample and 6 samples/frame.

FIG. 26 is a schematic view of an exemplary compact CMOS mixer circuitsuitable for pixel implementation in the system of FIG. 1.

FIG. 27 is a graph illustrating an exemplary excitation for each dye andlaser.

FIG. 28 is a schematic representation of an analytic device suitable foruse in the system of FIG. 1.

FIG. 29 is a schematic representation of the operation of an exemplarydetector suitable for use in the system of FIG. 1.

FIG. 30 is a schematic representation of an exemplary analytic devicesuitable for use in the system of FIG. 1.

FIG. 31 is a schematic representation of an analytic device convertingemission profiles suitable for use in the system of FIG. 1.

FIG. 32 is a schematic representation of an analytic device convertingemission profiles suitable for use in the system of FIG. 1.

FIG. 33 is a schematic representation of an analytic device convertingemission profiles suitable for use in the system of FIG. 1.

FIG. 34 is a schematic representation of an analytic device convertingemission profiles suitable for use in the system of FIG. 1.

FIG. 35 is a schematic representation of an analytic device convertingemission profiles suitable for use in the system of FIG. 1.

FIG. 36 is a graph illustrating the response of exemplary fluorophoresto a stimulus.

FIG. 37 is a schematic representation of a photodetector with a driftregion and storage suitable for use in the system of FIG. 1.

FIG. 38 is a schematic representation of a stimulus temporally filteredfrom a fluorophore decay signal in the drift region suitable for use inthe system of FIG. 1.

FIG. 39 is a schematic representation of a basic single pulse operationof a drift optode suitable for use in the system of FIG. 1.

FIG. 40 is a graph illustrating the decay response for differentfluorophore combinations.

FIG. 41( a) to (d) is a schematic sequence of illustrating an exemplarymethod of fabricating an analytic device suitable for use in the systemof FIG. 1

DETAILED DESCRIPTION

Reference will now be made in detail to various embodiments of thepresent invention(s), examples of which are illustrated in theaccompanying drawings and described below. While the invention(s) willbe described in conjunction with exemplary embodiments, it will beunderstood that present description is not intended to limit theinvention(s) to those exemplary embodiments. On the contrary, theinvention(s) is/are intended to cover not only the exemplaryembodiments, but also various alternatives, modifications, equivalentsand other embodiments, which may be included within the spirit and scopeof the invention as defined by the appended claims.

The present invention is generally directed to improved systems, methodsand devices for use in optical analyses, and particularly, opticalanalyses of biological and/or chemical samples and reactions.

Various aspects of the systems and methods described herein are similarto those described in U.S. Provisional Patent Application No. 61/306,235entitled INTEGRATED ANALYTICAL DEVICES AND SYSTEMS, U.S. ProvisionalPatent Application No. 61/387,916, entitled INTEGRATED ANALYTICAL SYSTEMAND METHOD, U.S. Provisional Patent Application No. 61/410,189, entitledILLUMINATION OF INTEGRATED ANALYTICAL SYSTEMS, the entire content ofwhich is incorporated herein for all purposes by this reference.

In various respects, the optical analyses of the invention generallyseek to gather and detect one or more optical signals, the appearance ordisappearance of which, or localization of which, is indicative of agiven chemical or biological reaction and/or the presence or absence ofa given substance within a sample material. In some cases, thereactants, their products, or substance of interest (all of which arereferred to as reactants herein) inherently present an opticallydetectable signal which can be detected. In other cases, reactants areprovided with exogenous labeling groups to facilitate their detection.Useful labeling groups include fluorescent labels, luminescent labels,mass labels, light scattering labels, electrochemical labels (e.g.,carrying large charge groups), metal labels, and the like. Exemplars ofsuch labeling groups are disclosed by U.S. Pat. No. 7,332,284 and U.S.Patent Application Publication Nos. US 2009/0233302 A1, US 2008/0241866A1, and US 2010/0167299 A1, the entire content of which is incorporatedherein for all purposes by this reference.

In various embodiments, one or more reactants in an analysis is providedwith a fluorescent labeling group that possesses a fluorescent emissionspectrum that is shifted from its excitation spectrum, allowingdiscrimination between the excitation light source and the emission ofthe label group. These fluorescent labels typically have high quantumyields, further enhancing their detectability. A variety of differentfluorescent label groups are well known in the art, and includefluorescein and rhodamine based organic dyes, such as those sold underthe Cy3 and Cy5 labels from, e.g., GE Healthcare, and the AlexaFluor®dyes available from Life Technologies, Inc. A wide variety of organicdye structures have been previously described in the art.

Other fluorescent label groups include, for example, particle-basedlabeling groups. Some such particle label groups constitute encapsulatedor otherwise entrained organic fluorophores, while others comprisefluorescent nanoparticles, such as inorganic semiconductor nanocrystals,e.g., as described in U.S. Pat. Nos. 6,207,392, 6,225,198, 6,251,303,6,501,091, and 7,566,476, the entire content of which is incorporatedherein for all purposes by this reference.

By detecting these fluorescent labeling groups, one can ascertain thelocalization of a given labeled reactant, or detect reaction events thatresult in changes in the spectral or other aspects of the fluorescentlylabeled reactant. For example, in binding or hybridization reactions,the ability of a labeled reactant to bind to another immobilizedreactant is detected by contacting the reactants, washing unboundlabeled reactant away, and observing the immobilized reactant to lookfor the presence of bound fluorescent label. Such assays are routinelyemployed in hybridization assays, antibody assays, and a variety ofother analyses.

In a number of different nucleic acid sequencing analyses,fluorescently-labeled nucleotides are used to monitor thepolymerase-mediated, template-dependent incorporation of nucleotides ina primer extension reaction. In particular, a labeled nucleotide isintroduced to a primer template polymerase complex, and incorporation ofthe labeled nucleotide is detected. If a labeled nucleotide isincorporated, it is indicative of the underlying and complementarynucleotide in the sequence of the template molecule. In traditionalSanger sequencing processes, the detection of incorporation of labelednucleotides utilizes a termination reaction where the labelednucleotides carry a terminating group that blocks further extension ofthe primer. By mixing the labeled terminated nucleotides with unlabelednative nucleotides, one generates nested sets of fragments thatterminate at different nucleotides. These fragments are then separatedby capillary electrophoresis, to separate those fragments that differ bya single nucleotide, and the labels for the fragments are read in orderof increasing fragment size to provide the sequence (as provided by thelast-added, labeled terminated nucleotide). By providing a differentfluorescent label on each of the types of nucleotides that are added,one can readily differentiate the different nucleotides in the sequence(see, e.g., U.S. Pat. No. 5,821,058, the entire content of which isincorporated herein for all purposes by this reference).

In newer generation sequencing technologies, arrays of primer-templatecomplexes are immobilized on surfaces of substrates such that individualmolecules or individual and homogeneous groups of molecules arespatially discrete from other individual molecules or groups ofmolecules, respectively. Labeled nucleotides are added in a manner thatresults in a single nucleotide being added to each individual moleculeor group of molecules. Following the addition of the nucleotide, thelabeled addition is detected and identified.

In some cases, the processes utilize the addition of a single type ofnucleotide at a time, followed by a washing step. The labelednucleotides that are added are then detected, their labels removed, andthe process repeated with a different nucleotide type. Sequences ofindividual template sequences are determined by the order of appearanceof the labels at given locations on the substrate.

In other similar cases, the immobilized complexes are contacted with allfour types of labeled nucleotides where each type bears adistinguishable fluorescent label and a terminator group that preventsthe addition of more than one nucleotide in a given step. Following thesingle incorporation in each individual template sequence (or group oftemplate sequences,) the unbound nucleotides are washed away, and theimmobilized complexes are scanned to identify which nucleotide was addedat each location. Repeating the process yields sequence information ofeach of the template sequences. In other cases, more than four types oflabeled nucleotides are utilized.

In particularly elegant approaches, labeled nucleotides are detectedduring the incorporation process, in real time, by individual molecularcomplexes. Such methods are described, for example, in U.S. Pat. No.7,056,661, the entire content of which is incorporated herein for allpurposes by this reference. In these processes, nucleotides are labeledon a terminal phosphate group that is released during the incorporationprocess so as to avoid accumulation of label on the extension productand avoid any need for label removal processes that can be deleteriousto the complexes. Primer/template polymerase complexes are observedduring the polymerization process, and nucleotides being added aredetected by virtue of their associated labels. In one particular aspect,they are observed using an optically confined structure, such as a zeromode waveguide (see, e.g., U.S. Pat. No. 6,917,726, the entire contentsof which is incorporated herein for all purposes by this reference) thatlimits exposure of the excitation radiation to the volume immediatelysurrounding an individual complex. As a result, only labeled nucleotidesthat are in the process of being incorporated are exposed to excitationillumination for a time that is sufficient to identify the nucleotide.

In another approach, the label on the nucleotide is configured tointeract with a complementary group on or near the complex, e.g.,attached to the polymerase, where the interaction provides a uniquesignal. For example, a polymerase may be provided with a donorfluorophore that is excited at a first wavelength and emits at a secondwavelength, while the nucleotide to be added is labeled with afluorophore that is excited at the second wavelength but emits at athird wavelength (see, e.g., the above-mentioned '661 patent). As aresult, when the nucleotide and polymerase are sufficiently proximal toeach other to permit energy transfer from the donor fluorophore to thelabel on the nucleotide, a distinctive signal is produced. Again, inthese cases, the various types of nucleotides are provided withdistinctive fluorescent labels that permit their identification by thespectral or other fluorescent signature of their labels.

As will be appreciated, a wide variety of analytical operations may beperformed using the overall reaction framework described herein and areapplicable to the present invention. Such reactions include reactiveassays, e.g., examining the combination of reactants to monitor the rateof production of a product or consumption of a reagent, such as enzymereactions, catalyst reactions, etc. Likewise, associative or bindingreactions may be monitored where one is looking for specific associationbetween two or more reactants, such as nucleic acid hybridizationassays, antibody/antigen assays, coupling or cleavage assays, and thelike.

The analytical system in accordance with the present invention employsone or more analytical devices referred to as “optode” elements. In anexemplary embodiment, the system includes an array of analytical devicesformed as a single integrated device. An exemplar of a suitable optodeelement is disclosed by the above-mentioned '235 application. Theexemplary array is configured for single use as a consumable. In variousembodiments, the optode element includes other components including, butnot limited to, local fluidics, electrical connections, a power source,illumination elements, detector elements, logic, and a processingcircuit. Each analytical device or array is configured for performing ananalytical operation as described above.

Turning now to the drawings, wherein like components are designated bylike reference numerals throughout the various figures, attention isdirected to FIG. 1 which shows a optics collection and detection system,generally designated 30 which generally includes a reaction cell 32, inwhich the reactants are disposed and from which the detector opticalsignals emanate.

“Reaction cell” is to be understood as generally used in the analyticaland chemical arts and refers to the location where the reaction ofinterest is occurring. Thus, “reaction cell” may include a fullyself-contained reaction well, vessel, flow cell, chamber, or the like,e.g., enclosed by one or more structural barriers, walls, lids, etc., orit may comprise a particular region on a substrate and/or within a givenreaction well, vessel, flow cell or the like, e.g., without structuralconfinement or containment between adjacent reaction cells. The reactioncell may include structural elements to enhance the reaction or itsanalysis, such as optical confinement structures, nanowells, posts,surface treatments such as hydrophobic or hydrophilic regions, bindingregions, or the like. The reaction cell can comprise a nanoscale featuresuch as a nanoscale well, and can comprise a zero mode waveguide (ZMW)as described in U.S. Pat. No. 7,315,019, the entire content of which isincorporated by reference herein for all purposes.

In various respects, “analytical device” refers to a reaction cell andassociated components that are functionally connected. In variousrespects, “analytical system” refers to one more associated analyticaldevices and associated components. In various respects, “analyticalsystem” refers to the larger system including the analytical system andother off-chip instruments for performing an analysis operation such asa power source and reservoir.

In some cases, one or more reactants for the reaction of interest may beimmobilized, entrained or otherwise localized within a given reactioncell. A wide variety of techniques are available for localization and/orimmobilization of reactants including surface immobilization throughcovalent or non-covalent attachment, bead, or particle-basedimmobilization, followed by localization of the bead or particle,entrainment in a matrix at a given location, and the like. Reactioncells may include ensembles of molecules, such as solutions or patchesof molecules, or it may include individual molecular reaction complexes,e.g., one molecule involved in the reaction of interest as a complex.Similarly, the overall devices and systems of the invention may includeindividual reaction cells or may comprise collections, arrays, or othergroupings of reaction cells in an integrated structure, e.g., amultiwell or multi-cell plate, chip, substrate or system. Some examplesof such arrayed reaction cells include nucleic acid array chips, e.g.,GeneChip® arrays (Affymetrix, Inc.), zero mode waveguide arrays (asdescribed elsewhere herein), microwell and nanowell plates, multichannelmicrofluidic devices, e.g., LabChip® devices (Caliper Life Sciences,Inc.), and any of a variety of other reaction cells. In variousrespects, the “reaction cell”, sequencing layer, and zero modewaveguides are similar to those described in U.S. Pat. No. 7,486,865,the entire content of which is incorporated herein for all purposes bythis reference.

Although the exemplary analytical system includes an array of analyticaldevices having a single waveguide layer and reaction cell layer, onewill appreciate that a wide variety of layer compositions may beemployed in the waveguide array substrate and cladding/reaction celllayer and still achieve the goals of the invention (e.g., U.S. PatentApplication Publication No. US 2008/0128627 A1, the entire content ofwhich is incorporated herein for all purposes by this reference).

With continued reference to FIG. 1, analysis system 30 typicallyincludes one or more analytical devices 33 having a detector 35, whichis disposed in optical communication with a respective reaction cell 32,that is, the detector element is configured for direct detection of anemission event within respective reaction cell. One will appreciate thatsuch optical communication may include radiative or non-radiativecommunication. Optical communication between the reaction cell and thedetector element may be provided by an optical train or channel 37 whichmay include one or more optical elements for efficiently directing thesignal from reaction cell 32 to detector 35. These optical elements maygenerally comprise any number of elements, such as lenses, filters,gratings, mirrors, prisms, refractive material, or the like, or variouscombinations of these, depending upon the specifics of the application.The reagents can be supplied to the reaction cells via a fluid portwhich is connected to the reaction cells through fluid conduits 40.Typically, the analytical reaction that is measured within the reactioncell can be monitored using fluorescence. The illumination (excitation)light for such fluorescence can be supplied via a light pipe as shown inFIG. 1, via a waveguide 39. The fluid port and light pipe can each beused to address multiple analytical devices or elements at one time.

In various embodiments, the reaction cell and detector element areprovided along with one or more optical elements in an integrated devicestructure, such as that shown in FIG. 2. By integrating these elementsinto a single device architecture, one improves the efficiency of theoptical coupling between the reaction cell and the detector. Inparticular, in conventional optical analysis systems, discrete reactionvessels are typically placed into optical instruments that utilizefree-space optics to convey the optical signals to and from the reactionvessel and to the detector. These free space optics tend to includehigher mass and volume components, and have free space interfaces thatcontribute to a number of weaknesses for such systems. For example, suchsystems have a propensity for greater losses given the introduction ofunwanted leakage paths from these higher mass components, and typicallyintroduce higher levels of auto-fluorescence, all of which reduce thesignal-to-noise ratio (SNR) of the system and reduce its overallsensitivity, which in turn can impact the speed and throughput of thesystem. Additionally, in multiplexed applications, signals from multiplereaction regions (i.e., multiple reaction cells or multiple reactionlocations within individual cells) are typically passed through a commonoptical train, or common portions of an optical train, using the fullvolume of the optical elements in that train to be imaged onto thedetector plane. As a result, the presence of optical aberrations inthese optical components, such as diffraction, scattering, astigmatism,and coma, degrade the signal in both amplitude and across the field ofview resulting in greater noise contributions and cross talk amongdetected signals. Furthermore, these free-space optical systems can belarge and bulky.

The devices of the invention, in contrast, include relatively lowvolumes between the reaction cell and the detector, thereby reducing thenoise contributions from those components and provide few or no freespace interfaces that can contribute to the noise profile of the systemthrough the introduced reflections and losses from large index changesfrom the components to air or free space. Further, in preferred aspects,a given reaction region is provided with its own devoted optical trainto direct signals to a devoted portion of the sensor. In addition theintegrated systems described herein can be made small and compact.

In various embodiments, the device is configured such that emitted lightfrom the fluorescent species in the nanoscale well is not transmittedthrough free space. By “not transmitted through free space” it isgenerally meant that the respective element (e.g. a signal or energy) isnot transmitted through open space or free space optics, and in variousrespects, is transmitted only through light-modulating media. Suchlight-modulating media may include refractive devices such as an opticallens that makes use of the refractive index of a defined volume of air.By contrast, light-modulating media generally does not include ambientair open to the environment.

As a result of the integrated architecture, optical aberrations areconfined to individual reaction regions as opposed to being appliedacross an entire array of reaction regions. Likewise, in a furtheraspect, the reaction region, optical train, and detector are fabricatedin an integrated process, e.g., micromechanical lithographic fabricationprocesses so that the components are, by virtue of the manufacturingprocess, pre-aligned and locked into such alignment by virtue of thefabrication process. Such alignment is increasingly difficult using freespace optics systems as reaction region sizes decrease and multiplexincreases. In addition, by integrating such components into one unifiedcomponent, relative movement between such sub-components, as is the casewith free space optics, can make drift and continued alignment resultingfrom vibrations a more difficult task. Likewise, the potential forcontamination in any of the intermediate spaces (e.g. dust and othercontaminants), is eliminated (or at least substantially reduced) in anintegrated system as compared to free space systems.

In addition to reducing noise contributions from the optical pathway,the integrated devices of the invention also benefit from fabricationprocesses and technology that eliminate other issues associated withdiscrete reaction cell, optic, and detection components. For example,with respect to certain highly multiplexed or arrayed reaction cells,initial alignment and maintaining alignment of the detection with thereaction cell over the full length of the analysis can raisedifficulties. This is particularly the case where excitationillumination may be specifically targeted among different arraylocations of the reaction cell and/or among different reaction cells.

As used herein, the term “integrated” may have different meanings whenused to refer to different aspects of the invention. For example, in thecase of an integrated device or an integrated optical system, the term“integrated” generally means that the various components are physicallyconnected, and that the optical signals pass from component to componentwithout passing into air or free space, as would be understood by one inthe field of optics. In the context of the description of a system, theterm “integrated” is to be understood as generally used in theanalytical and electrical engineering fields, where “integrated” wouldrefer, for example, to a combination or coordination of otherwisedifferent elements to provide a harmonious and interrelated whole,whether physically or functionally. The meaning of the term willgenerally be understood by one of skill in the art by the context inwhich it is used.

With continued reference to FIG. 2, being an integrated device, thelight emitted from the reactor cell 32 will pass through to detector 35without passing through air or free space. In some embodiments, theintegrated analytical device also comprises components for providingillumination to the reactor cell. For example, in many cases where thereactor cell includes a zero mode waveguide, it is often desirable toprovide illumination from below the reactor cell, for example betweenthe bottom of reactor cell and the transmission layer or optical train37. In some cases, a waveguide 39 is incorporated into the analyticaldevice to provide such illumination. Analytical devices comprisingwaveguides for illumination are described in more detail herein, and forexample, in U.S. Pat. No. 7,820,983 and U.S. Patent ApplicationPublication No. US 2010/0065726 A1, the entire content of which isincorporated herein for all purposes by this reference.

In various embodiments, the analytical device is a substrate including areaction cell array, and a detector array on a bottom surface of thearray. The device may also include other components such as processingcircuits, optical guides, and processing circuits. In variousembodiments, the analytical device may be formed by building layers on asubstrate or by bonding two or more substrates. In an exemplary device,a fused silicon (FuSi) substrate, a zero-mode waveguide (ZMW) layer, anda silicon substrate with a photodetector array are bonded together toform the array of analytical devices, such as the ones described in theabove-mentioned '189 and '916 applications. One will appreciate thatsuch integrated analytical devices have significant advantages in termsof alignment and light collection. For example, the reaction site anddetector are aligned through the manufacturing process. One willappreciate from the description herein, that any of the components andsystems may be integrated or modified in various ways. In anotherexample, the ZMW substrate and detector array are on separate substratesthat are brought together for the experiment, after which the ZMWsubstrate is replaced with another substrate for a second experiment.With this approach, the detector array may be re-used rather than beingdisposed with the ZMW substrate after an experiment. It may also be morecost effective as the yields from each of the processes are separated.In this manner, the ZMW array and detector array are in intimate contactduring the experiment (as if they are part of an integrated device), butthey can be separated after the measurement.

The size of the processing circuits in each of the analytical devicesmay be minimized to reduce costs. By developing a board in the receivercamera electronics (e.g. massively parallel DSP or microprocessor or adedicated FPGA, CPLD or ASIC), overall operating costs (i.e.$/mega-base) may be minimized.

With further reference to FIG. 2, analytical device 33 has a reactioncell 32 that is coupled with a reagent reservoir by fluid conduit 40which delivers reactants to the reaction cell. The reaction cell can bea nanoscale well or zero mode waveguide. In some cases, the reactioncell will have a biomolecule such as a polymerase enzyme immobilizedwithin it. The fluidic conduit can provide reagents across a number ofreaction cells. Below the reaction cell is a waveguide for providingexcitation illumination to the reagents within the reaction cell. Whilea waveguide is shown here, other optical elements such as those providedelsewhere herein can be used to provide light from under the reactioncell. The illumination light can be used to excite fluorescent emissionfrom reagents with the reactor cell. The light emitted from the reactioncell is directed downward through a transmission layer, which acts totransmit the light from the reaction cell to the detector. In somecases, the transmission layer will have optical components to enhancethe efficiency of the light transfer or modulate the light.

In the analytical device of FIG. 2, optical conduit or optical train 37is in optical communication with reaction cell 32, and is in turn inoptical communication with detector 35. In some cases, the detector hasmultiple sensing elements, each for detecting light having a differentcolor spectrum. For example, in the case of sequencing, the detector foreach reaction cell may have four elements, one for each of the fourbases. In some cases the sensor elements provide color discrimination,in other cases, color filters are used to direct the appropriate colorof light to the appropriate sensor element shown as a multicolordiscriminating set of sensor elements 35 w-35 z. In the illustratedembodiment, four sensor elements are shown (e.g., 35 w, 35×, 35 _(y),and 35 _(z)), however, one will appreciate that one, two, three, four ormore sensors may be utilized. The sensor elements are coupled toappropriate electronic components 44, such as busses and interconnects,that make up the overall sensor or camera. The electronic components canalso include processing elements for processing the signal from thedetectors.

One aspect of the invention is an integrated sensor element that iscapable of discriminating signal from multiple fluorophores as afunction of time in real time. These types of systems can be used formonitoring analytical chemical and biochemical reactions such as theaction of a polymerase in order to carry out nucleic acid sequencing asdescribed above. Several approaches for monitoring multiple fluorophoresfrom one sensor element are described herein. In some embodiments, thedifferent fluorophores are detected at spatially different locations. Insome embodiments, each different fluorophore is detected by a singledetector element, for example using a pixel which can discriminatedifferent colors of light by the depth at which the light penetratesprior to detection. In some embodiments, the different fluorophores arediscriminated by their different fluorescent decay profiles (lifetime).In yet other embodiments, the different fluorophores are excited bydifferent illumination sources, each of which is modulated with time,and the identity of the fluorophore is identified by correlating thetiming of the emission with the timing of the excitation. In some cases,these methods for discriminating the fluorophores can be combined forimproving detection and discrimination.

Photonic Band Gap Detector

The excitation/detection components of current optical collectionsystems are generally very complex with numerous lenses, dichroics,holographic phase masks and other elements that are expensive, difficultto keep in proper alignment and prone to increased downtime due to theircomplexity.

In some embodiments, the system of the present invention combines thewaveguide illumination of the reaction cell with a set of photonic bandgap (PBG) layers that take the place of dichroic filters in currentsystems, and one to four detectors per reaction cell, and a unifiedsolid state device can be achieved. This PGB approach provides onemethod of spatially discriminating different fluorophores.

With reference to FIG. 3, each reaction cell 32 is provided with anoptic train 37 including at least one channel having several layers ofPBG layers 51 which are configured to initially reject the pump laser,that is, to reject the illumination light (λ₁) provided by illuminatingwaveguide 39 but allow excitation light to selectively pass through eachchannel to a respective detector (e.g., 35, 35 _(x)). For example, theillustrated embodiment includes four channels (two of which are shown inFIG. 3, all of which are shown in FIG. 4A), each of which are opticallyisolated from one another by a metal shield or other suitable cladding47 so that excitation light may be directed to a respective sensorelement (e.g., 35, 35 _(x)).

The top layer contains reaction cell 32 with an illuminating waveguide39 below. As illustrated, a device channel may be created with a seriesof dielectric posts 49 with high index of refraction, which postsprovide lateral confinement of pump laser light from the illuminatingwaveguide to the region about the reaction cell, and may allow higherlight density than a simple fused silica layer without PBG layers forthe same pump intensity.

Even though the index mismatch of the waveguide and cladding may preventmost light from escaping towards the sensor (by total internalreflection), a one-dimensional (1-D) PBG layer 51 that passes emissionwavelengths but attenuates pump lines may be added for better SNR. Forexample, the 1-D PBG layer may reject λ₁ but allow λ₂ and λ₃ to pass tothe photodetectors below. The 1-D PGB layer may include one or moresemiconductor-based structures of miniature band-stop filters. PBGfilters are especially suitable for use with the systems and devices ofthe present invention as they are much easier to manufacture and usewith other circuits, and is far more compact than conventional filters.

In the next level down, two more PBG stacks 53 are shown side-by-side,which pass different wavelengths, hence create wavelength specificity.These are essentially band-pass filters. The illustrated embodiment is afour color system including the two stacks shown in FIG. 3 (e.g., stacks53, 53 _(x)), and two more stacks with different spectral properties aredirectly behind. In the illustrated embodiment, the PBG stacks areconfigured to pass different excitation light of different wavelengthsin the range of approximately 100 nm to 1 μm. For example, stack 53 maybe configured to allow excitation light of wavelength λ₂ to pass butreject light of wavelength λ₃, while stack λ_(x) may be configured toallow excitation light of wavelength λ₃ to pass but reject light ofwavelength λ₂. The illustrated embodiment includes four stacks, which invarious embodiments may be sufficient to pass sufficient light with outfurther guides, however, one will appreciate that one, two, three, fouror more PBG stacks may be utilized to provide the desired number ofchannels.

Optionally, metal attenuators 47 or other suitable cladding may be addedadjacent to the stacks to prevent crosstalk, or vertically-oriented 1-Dbroadband PBG layers can be added that reflect and guide light towardthe respective sensor elements 35, 35 _(x) of the detector.

Turning now to FIG. 4A, detector 35 is may be a quad-photo-diode (QPD)detector having electrically separated quadrants serving as each sensorelement (e.g., 35, 35 _(x), 35 _(y) and 35 _(z)) which measuresrespective signals. Preferably, each sensor element is aligned directlybelow a corresponding PBG stack (e.g., 53, 53 _(x)) thereby formingrespective optic channels. Each sensor element may determine whenexcitation light passes through a respective PBG stack (e.g., 53, 53_(x), etc.).

Alternatively, the detector may be a position sensitive diode such as aPIN diode junction that provides the relative location of the eachphoton pulse as it lands on the detector. For example, detector 35 a mayhave a generally square configuration including a relatively thickintrinsic semiconductor (I) between an upper p-type semiconductor (P)and n-type semiconductor regions (N) positioned at the four lowercorners of the detector.

Depending upon the proximity to the respective corner n-type regions(e.g., I₁, I₂, I₃, I₄), one may determine through which PBG stack (e.g.,53, 53 _(x), etc.) excitation light passes.

Alternatively, a Bragg filter/dielectric stack/PBG can be used with asingle mode fiber and can be used to filter out (e.g., reflect) theinput light from the laser while letting the emission light through tothe detector. One will appreciate that ZMW emissions have a widedistribution of angles, and that multi-layer dielectric filters cantolerate only a narrow distribution of angles. The fluorescent dipole isvery small and thus the emissions occupy a very small phase spacevolume, and it may be efficiently coupled into a single-mode waveguide,and grating filters can be implemented in single-mode filters.Accordingly, a coupling between a ZMW and a single-mode waveguide mayinclude an in-line Bragg filter to screen out the excitation light.

For example, a Bragg grating between the ZMW and the detector may simplyacts as an interference element to make spatially unique patterns on a1D or 2D detector, in a manner similar to that shown in FIG. 34(discussed below), which assumes a point source and the optical pathlength variation to cause the interference. One would appreciate thatone generally wants collimated light at the grating, and thus amicrolens may be provided to direct light from the ZMW to the Bragggrating, and in turn, the detector. Optionally, the Bragg grating mightbe at an angle so that the 00 pump spot is dumped off the detector arrayand only higher order peaks of the pump are measured and all orders ofthe dyes.

Integrated Readout Buses, Detector Absorption, etc.

Integrated sequencing systems and devices require compact and efficientillumination methods to operate at high resolution for low costsequencing. One limitation is the rejection of source illumination. Toeffectively reject the source illumination, a method for filtering thefrequencies of the source and passing the frequencies of the excitationlight or emission need to be incorporated. Various aspects of thepresent invention relate to methods of using the detector opticalproperties to perform this filtering to enable compact and low costsensors.

Various aspects of integrated sequencing sensors utilizing illuminationconfinement to small molecular volumes has been described in theabove-mentioned '235 and '916 applications. A cross-section of anexemplary integrated device 33 c is shown in FIG. 5 and includes asubstrate 54 that may be provided with patterned photodetectors 35 cwhich can be single devices or an array of 1 or 2 dimensions ofarbitrary resolution. These photodetectors are arranged with conductivereadout busses 56 which generally are opaque to radiation. These metalconductors are arranged in an insulating layer. In the illustratedembodiment, the insulating layer is made from an oxide dielectric andtransmissive to optical radiation frequencies and may function as theilluminating waveguide 39 c. Such an integrated device may contain asequencing layer which is made from very small volume optical cavitiesor reaction cells 32 c arranged over the photodetectors 35 c. Thesereaction cells may be made within a top layer 58 which is generallyopaque and biocompatible with any required surface treatments tofacilitate the sequencing chemistries.

The detector layer has a spectral range where it can operate. As anexample, the optical absorption coefficient for silicon is shown in FIG.6. This represents the coefficient in the exponential decay of normaldirected incident photons at each wavelength into the material. It iseasily shown from this curve that longer wavelength photons can passthrough the material if the material thickness is thin enough. A sourceof radiation may be chosen to match with an engineered materialthickness of substrate to be fully transmitted through the photodetector and substrate. If a standard fluoroscopic dye material is usedand the emission and excitation frequencies are carefully chosen tostraddle the absorption profile of this engineered wafer, the shortwavelength tail of the emission distribution could fall within thedetector absorption band while the excitation frequency can be placed inthe transmissive region. This is shown in FIG. 7 using the siliconabsorption curve as example.

The effectiveness of this approach is increased by maximizing the Stokesshift of the dye and operating in a region where the thickness of thewafer can be manufacturable. For a silicon based detector, this is inthe 1100-1200 nm region.

An alternative to wafer thickness tuning of the transmission edge is touse a buried detector or a back illuminated detector with a thicksubstrate. Another alternative is to use a signal collection photodiodeon the front surface and an emission drain photodiode on the backsurface. In this approach, the deeper penetrating source photons may becaptured and drained to the external circuit while the less penetratingsignal photons are captured by the front surface detector and stored.

Doped Fused Silica, Microprism

Dispersion prisms were critical to the performance many existingsystems, while dichroic optics are increasingly used for wavelengthselectivity. Various aspects of the present invention allow for theintegration of dispersion optics into the a semiconductor chip,preferably combining reaction cell with the dispersive element(s).Dispersive elements allow for the spatial separation of differentwavelengths of light, allowing for the detection of more than onefluorophore in one optical analysis element.

With reference to FIG. 5, various components of system 30 c may beintegrated into a semiconductor chip. For example, reaction cell 32 c,detector 35 c, illuminating waveguide 39 c and electronic components(e.g., readout busses 56 may be formed directly on substrate 54, variousaspects of which will be discussed in further detail below.

In accordance with the present invention, heavy metals or semiconductorsmay be doped into fused silica or standard silicon oxide to create anindex of refraction gradient, essentially creating a microprism 61, asshown in FIG. 8. This doped region, mounted near a reaction cell woulddisperse the light at different angles by wavelength, essentiallyconverting the spectral difference to an angular difference (or in theFourier plane, a transition). Generally, this device would be on theorder of a couple of microns dimensionally.

Optionally, a collimator 63 may be provided adjacent the reaction cell32 a micromirror or a micromirror be fabricated adjacent to or under thereaction cell collimate the outgoing beam and simplify the action of themicroprism to be acting on parallel rays.

Various dopants, gradients, size ranges (nanometers to microns), as wellas various structures (e.g., various micromirror configurations,microlens configurations) may be utilized with the present invention.One will also appreciate that laser illumination may be provided via anillumination waveguide above microprism or through chip.

Processes for Producing Filters

Other aspects of the present invention are directed to the use ofmulti-layer optical filters over photodetectors for improved spectralselection and rejection. In particular, a compact molecular sequencingdetection system can benefit from the rejection of source excitationradiation that would reduce the sensitivity of the fluorophore signal.The effectiveness of the filter is dependent on the incident angle ofthe illumination.

Described herein are methods to pattern multi-layer filters below thesurface, as well as methods to pattern these materials on a non-planarsurface to reduce the distribution of incident angles from a wide angleillumination source.

Methods for the patterning of thin films for optical filtering onsemiconductor substrates is well known in the art. Techniques forpatterning these films at the atomic level are performed on commercialdevices. Multi-layer devices are effective at narrow band rejection ortransmission of light. In particular, dichroic filters are important asthey pass a narrow band of light while reflecting the out of bandwavelengths. These filters do not absorb out of band spectra and do notheat as much. They are however very sensitive to incident anglevariations.

In the case of local dye emitters located close to the photodetectors,the emission profile is generally broad in solid angle extent (up toLambertian). By placing a hemispherically patterned optical filter inproximity to the source (prior to significant scattering or reflection),each ray of light will he normally incident on the filter surface. Bypatterning hemispherical filters near these emitters, high performancefilters can be utilized for high performance sensing applications likesingle molecule spectroscopy without the need for large free-spaceoptics.

FIG. 9( a) to FIG. 9( m) illustrate a set of semiconductor processingsteps enable this device. The set of filters is patterned on the surfaceof substrate 54, which may be a 6″ fused silica substrate (see FIG. 9(a)). A sacrificial deposition (SD) is applied, for example by aTi/Chrome/DC sputter in an otherwise conventional fashion (see FIG. 9(b)), and a poly etch back and surface clean may then be applied is anotherwise conventional fashion (see FIG. 9(c)). Non critical lithographymay be used to form a protective dome (PD) to protect areas of thesubstrate to remain intact (see FIG. 9( d)). An anisotropic silica etchis performed which leaves the protected areas intact and etches theunprotected areas with significant sidewall ingress (see FIG. 9( e)) toform hemispherical cavities. One will appreciate that various suitableanisotropic etch chemistries may be used, and may be wet or dry tocreate the desired shape.

The mask may then be removed by conventional ash and clean techniques(see FIG. 9( f)), and the thin film filter is processed with standardvapor deposition techniques to provide a combination thin filmdeposition for filter. Conventional PVD/CVD techniques may be used todeposit a thin film (TF)(see FIG. 9( g)). Conventional lithographytechniques may again be used to provide protection within thehemispheres formed.

The filters within the hemispheres are protected as the filter materialremoved from the top silica surface (see FIG. 9( j)). An index matchedplanarization layer (PL) is deposited into the hemispheres for opticalperformance and surface planarization (see FIG. 9( k)). Theplanarization layer may be a hard layer such as PECVD oxide, ordepending on desired optical properties. A chemical polishing step isused for a clean flat surface (see FIG. 9( l)). One will appreciate thatconventional CMP or etch back techniques may be used to provide a flattopography. Follow on steps to complete the device can he made on thislayer. In particular, a reaction cell array (e.g., a ZMW array) can bepatterned with a metal pattern above this glass using proven processesto provide a top layer 58 (see FIG. 9( m)) into which the reaction cells32 may be formed (see FIG. 9( n)). One will appreciate that PVDdeposition may be used to deposit aluminum or other metals to form thetop layer.

It is important to note that the steps described above are compatiblewith follow-on steps following a conventional CMOS device fabrication.One will appreciate that this filter layer and ZMW device can be grownmonolithically above a CMOS imaging array to develop an integrated labon a chip biosensor in a standard commercial foundry.

Coupling of Fiber Optics

One problem that arises in collection systems for ZMW observation isthat it is difficult to raise the number of observed reaction cellsbeyond a limit imposed by available microscope objectives. Since theemission signal is weak it is necessary to collect over as large anumerical aperture as possible and then to relay the collected lightwith free-space optics to the camera pixel array with the appropriatemagnification, for example, approximately 60×. Objectives withmagnification and large numerical aperture (NA) have a limited field ofview and vice versa. There is an interrelationship between the lens NA,focal length (which relates to magnification), optical quality (whichrelates to the usable portion of the field of view) and the number ofresolvable points within its field of view.

One solution is to use a tapered fiber bundle array to help collect theemission from the reaction wells with a single fiber dedicated to eachsingle reaction cell. The most practical use of such an array would beto use a large field of view objective at a small magnification toconvey the light from the system chip to the input face of the fiberbundle. A minimum spacing for the fibers is around 3 microns. CurrentZMW spacing of the type described in the above mentioned '235 and '916applications is 1.33 microns so that only a magnification of 2.5 or 3×is necessary. Such a lens could cover a larger field of view thancurrent objectives. The individual fibers have large acceptancenumerical apertures near 1 and will therefore be efficient receivers ofthe light signal. The objective lens must also have a large NA so as notto lose light along the way, but since the fibers would act like lightbuckets imaging quality tolerances could be much looser than currentlyrequired. Once conveyed to the far end of the bundle the light would behandled in a conventional manner, for example, it may be separated intodifferent spectral components and imaged onto the camera pixels.

Because the fibers can be tapered up to a much larger size and/orspacing the magnification step is taken care of without the need forhigh tolerance optics and alignments. The size of the fibers at the farend can be chosen to optimize these steps. Another way to use the fiberbundle is to place the reaction cells directly on them. In this case thewhole tapered fiber bundle assembly becomes a part of the system chip.Alternatively, plates of fused fiber may be prepared where the fibersare oriented perpendicular to the plate faces. Such plates may be mademuch more economically. The fibers in these plates will act to conductthe reaction cell emission from one side to the other where a taperedfiber bundle could be positioned to further conduct the light to its farend where the individual ZMW signals are appropriately sized and spacedfor relay to the camera.

Because of the guided nature of the solution the form of the collectionarray at the reaction cells does not have to correspond to the form ofthe array imaged on the camera. For example a linear array of reactioncells could be reformed by appropriate arrangements of the fibers in thebundle into a 2D array, or a circularly symmetric pattern could bereformed into a rectangular array, etc. The light conducting elements donot have to be glass fibers but may be hollow tubes with reflectiveinternal surfaces.

Photodiode Surface Plasmon Resonance

Existing systems for optical detection of real-time single-molecule DNAsequencing have several other problems. First, multiplexing is partiallylimited by the physical limitations of optical resolution (which affectsthe density with which reaction wells can be placed on a chip and stillbe distinguished). Autofluorescence also limits multiplexing. Inaddition, when using fluorophores as the detection label, photodamage,may occur, affecting the achievable sequencing read-length. Finally,speed of data acquisition is often limited when using CCD cameras asdetectors, and it can be difficult to distinguish a true nucleotideincorporation from a branching event.

FIGS. 10A and 10B illustrate this aspect of the invention. In accordancewith the present invention, a polymerase 63 may be fixed to the detector35, for example, a photodiode in which the amount of current flowingthrough the device depends on the amount of incident light. In variousembodiments, the nucleotides may be labeled with a metal nanoparticle ora core-shell surface-enhanced Raman scattering particle 65, and for theincident illumination use a wavelength that is at or near the plasmonresonance of the nanoparticle. When the nucleotide (N) is incorporatedinto the polymerase, the nanoparticle will be very close (within severalnm) of the photodiode. A plasmon resonance will be excited in thenanoparticle, which will change the electromagnetic field in thevicinity of the photodiode. In this way, the presence of thenanoparticle will either enhance or reduce the baseline level ofphotocurrent in the photodiode (e.g., from original photocurrent I_(op)to differential photocurrent I_(op).

In various embodiments, one could use nanoparticles of different size,shape or material to distinguish between the four different nucleotides.Since there is no fluorescence involved, there would be less possiblephotodamage. Furthermore, the density of photodiode detectors would belimited by semiconductor fabrication methods rather than by opticalresolution, enabling greater multiplexing. Finally, data acquisitionfrom a photodiode may be faster than it is with a CCD camera, thusenabling fast polymerase dynamics to be captured more easily.

Four nucleotides could be distinguished by using differentsizes/shapes/compositions of metal nanoparticles or by using differenttypes of SERS-active nanoparticles. In addition, different excitationlasers that excite plasmons preferentially in certain nanoparticlescould be pulsed, and the detection of photocurrent in the photodiodecould be gated to these pulses.

One will appreciate that various types of nanoparticle may be used tolabel the respective nucleotides, various wavelengths of light may beemployed, various materials/structures may be used to fabricate thephotodiode, and/or various filtering coatings may be laid down on thephotodiode. In addition, since scattering is a coherent affect, theincident light may be modulated for heterodyne detection to decrease thebackground noise level.

Integrated Sensing and Reactor Cell

Other aspects of the present invention are directed to a design of anintegrated sensing and reaction cell where the contents of the emittedoptical signal are directed to the sensing element. As described above,the reaction cell is a chamber with extremely low volume as described inthe above-mentioned '215 and '916 applications, and known as a zero modewaveguide (ZMW). This reaction cell creates a stimulus illuminationvolume small enough to isolate a single nucleotide tagged with afluorophore during chemical incorporation. During this time, theillumination signal is emitted from reaction cell 32 in a Lambertiandistribution as shown in FIG. 11. To capture this light, reflective orrefractive elements capable of directing this light into a reduced solidangle or a detector capable of gathering the light over thishemispherical surface area can be incorporated to enhance the amount oflight that is captured.

A typical imaging pixel cross-section is shown in FIG. 12. A typicalCMOS pixel consists of a microlens, a color filter, dielectric stacks,metal interconnects and a photodiode. The photodiode element is locatedin the silicon substrate and is 4.5 microns below the top of the sensorsurface in this example. A microlens is added to many pixel designs todirect light towards the optically sensitive collection area of thepixel and away from the metal interconnects. Scattering and reflectioncan occur in the regions above the photodiode and the metalinterconnects disadvantageously reducing sensitivity.

In accordance with various aspects of the present invention, a pixeldesigned for intimate contact with the source signal is described. Themicrolens array is replaced with light guiding methods to confine anddirect all light coming from the wide distribution angles of the sourceto the photoactive area of the pixel. These approaches also reduce theleakage out of the pixel through reflection and scattering to otherpixels which would result in crosstalk. A method for fabricating the ZMWarrays directly on the unpassivated CMOS imaging array is presentedherein in addition to a method to align the two devices aftermanufacture. Utilizing a metal cladding tunnel from the nitridepassivation layer to the pixel for a reflective path as well as a totalinternal reflective path utilizing differences in refractive index isalso described.

With reference to FIG. 13, a design utilizing a metal tunnel createdabove photodiode 35 is created to reflect incoming light to thephotodiode is shown in a cross section. Metal interconnects and busses56 are located in the areas between pixels at lower metal layerscreating cones of in coming illumination that are funneled by the metal.At dimensions smaller than the wavelength of light, thetransverse-magnetic (TM) polarization at the metal surfaces attracts thelight to the metal dielectric interface forming a plasmon-polariton thatpropagates near the metal surface. This can lead to losses especially inless conductive metals (i.e. tungsten versus aluminum). Thetransverse-electric (TE) polarization efficiency is much higher.

One method of fabricating such cones utilizes planar metal layers andvias placed at a pitch much less than the wavelength of light, whichcreates a Faraday cage to confine electromagnetic (EM) waves within thecone, directing light to the detector as shown in FIG. 14A and FIG. 14B.

In various embodiments, a set of interconnected metal structures may beformed by the overlapping of a metal ring with an oxide step. In thisfashion detailed in FIG. 15( a) to (g), metal rings of increasing radiusare stacked on dielectric spacers to build the metal cone 67. The oxidesteps and metal thicknesses are designed to allow for full step coverageof the lattice. These same dielectric layers are utilized to insulatethe metal vias and busses (e.g., 56) on these same layers around theoptical cones. It is noted that photoresist patterning is designated“PP” in these Figures.

The use of a higher refractive index plug (such as silicon nitride(n=2.04) can be used to create a region with total internal reflection.When interfaced with silicon dioxide (n=1.46), total reflection occursfor angles less than 30°. The critical angle of reflection may becalculated from Snell 's Law.

θ_(C=cos) ⁻¹(n _(cladding) /n _(core))  Eq. (1)

In accordance with the present invention, a combination of upper levelmetal cladding and lower level nitride plugs can be used to direct lightto a pixel with high angles of incidence as shown in FIG. 16. The higherefficiency of coupling with nitride plugs can be used to advantage ifthe incident angles can be reduced from the wide angles output from thereactor chip. By designing the more lossy metal layers to direct lightto the lower layers and reduce the angles of incidence, this hybridapproach will yield higher efficiencies. In addition it is furtherdisclosed that high density bussing can be achieved by placing metallines adjacent to the nitride plug with reduced feature design rules.

Several approaches to an integrated monolithic manufacture of thereaction cell and pixel sensing cells may create a self containedstructure that contains all the emitted light to the cell. Threeexemplary fabrication methods are shown in FIG. 17( a) to (i).

In various embodiments, the reactor cell is grown on top of the imageroxide layer, as shown in FIG. 17( a) to (c). This is similar to theexisting fabrication techniques in which a thin layer of aluminum issputtered on a SiO2 substrate. Features are formed with a sub-micrometerimmersion lithography system (For example, like the ASML XM 1950) andetched. These low temperature processes can be performed after thestandard CMOS imaging device is fabricated.

In an alternative approach, the oxide above the top metal layer isremoved and the reactor aluminum layer is deposited directly on theremaining layers, as shown in FIG. 17( d) to (f). The pixel shield andreactor layer are conformally merged with the reactor aperture locatedwithin the flat oxide layer.

In another alternative approach, the surface of the CMOS sensor isplanarized using a standard chemical mechanical polish (CMP) step, asshown in FIG. 17( g) to (i). This exposes the top layers of the metalpixel shield with the SiO₂ optical pixel path in a flat plane. A ZMWmetal layer is sputtered onto this layer creating a self containedoptical cavity. The reactor aperture is patterned. The use of selfassembled multi-chip modules is also disclosed where known good die ofthe lower yielding component are impressed upon a wafer of the secondconstituent device.

Using one known approach, a silicon dioxide pattern is placed on the topsurface of the acceptor device, as shown in FIG. 18( a) to (c). Thislayer is hydrophilic and identified as “H” areas in the Figure.Contrasting areas of hydrophobic surrounding areas remain between andadjacent the “H” regions. One approach is in the preparation of anexcessively hydrophobic surface on the conductor and light shield bytreating an aluminum surface with nitric acid. The nitric acid treatmenthas been shown to alter the surface properties and increasehydrophobicity. After patterning these contrasting areas on bothacceptor and donor components, subsequent positioning these componentsin near alignment with a small amount of liquid shown in as gray ovals,surface tension self aligns these components. It has been shown thatalignment accuracy of less than 400 nm is possible and occurs within 100msec. The liquid evaporates and the devices are bonded with Van derWaals forces.

In various embodiments, the use of nanoscale optical spacers self alignto hydrophilic regions using surface tension in a liquid interface.These optical spacers can provide added optical distance, illuminationpathways, filtering, steering or focusing. This enhanced integratedphotonics assembly is shown in FIG. 19( a) to (d). An optical processinglayer may be comprised of individual elements or a patterned array ofoptical elements equivalent to the sensing and reacting pitch.

In these hybrid assemblies, with or without optical spacers, options forfully enclosed optical cavities are disclosed and illustrated in FIG.20( a) to (c). It should be noted that an illumination waveguide isadded to these figures. These waveguides are placed close to the zeromode waveguides to couple evanescent waves but isolated fromtransmission of this spatially decaying field from the sensing orreflective elements.

In FIG. 20( a), a discrete optical element used to focus energy to thepixel is shown. This element can be fabricated as a Fresnel, classical,spherical or holographic element with higher performance thantraditional photoresist based microlenses. This will direct energy froma high incident angle range onto the proximal pixel. The optical elementmay also contain antireflection or spectral filtering materials orlayers.

In FIG. 20( b), the optical spacer contains an optical aperture withreflective elements to contain the optical energy within the horizontalpixel extent. In addition, the use of a higher index material such assilicon nitride (shown in pink) may be patterned to enhance directedenergy to the lower pixel regions through total internal reflection. Inthis drawing, two example ray traces with reflections off the metal andnitride layers is shown to direct the energy downward to the pixel andcontain it within the horizontal pixel extent.

In FIG. 20( c), a zero mode waveguide made from optically transmissivematerial is shown. The evanescent wave is determined by the relativelocation of the illumination waveguide and the well. The addition of avery thin metal layer may also be added to enhance chemical performancewithout introducing significant optical effects (like scattering andplasmon resonance). To contain the emitted optical energy, the top layercontains reflective elements to redirect the energy within thehorizontal pixel extent. An optional spacer or focus element is shown asthe middle stacked layer that is aligned to continue optical reflectivepaths towards the photodetector.

Mulitplexed Homodyned and Heterodyned Pixels

Turning now to FIG. 21 and FIG. 22, the need to discriminate between thesignals of multiple tagged nucleotides with a single photo detector witha priori knowledge of the illumination wavelength energy and arrivaltime is addressed. This provides a means to incorporate a singlephotodetector to monitor the process of a chemical reaction withmultiple reagent tags as is done in the single-molecule real-time (SMRT)sequencing cell.

With a single photodetector, a savings in cost, complexity and size willbe realized. In one embodiment, an integrated pixel matched to the SMRTcell can be developed where the removal of free space optics willenhance photonic sensitivity. In the specific case where free spaceoptics is removed, reduced spectral effects for absorption, angularreflection to spectral distribution, etc. are reduced and theimplementation of a polychromatic stimulus is favorable for increaseddiscrimination.

A schematic of the system is shown in FIG. 21. In this system, an activeillumination source is used to stimulate the target. The target emissionand reflected stimulus signal is incident on an imaging array. Theimaging array contains a multitude of pixel sensing elements arranged ina repeating pattern to code the received image.

If the active imaging system is used with polychromatic multi spectralillumination, a system that can utilize this illumination is shown inFIG. 22. In this system, a controller is added that can sequence theillumination wavelength with varying power or pulse widths. Multiplewavelengths can be simultaneously activated or pulses can overlap or bediscretely enabled. Knowledge of the current or predicted illuminationof each wavelength can be provided to the imaging array. Alternatively,the illumination can be controlled based on feedback from the imagingarray.

A pixel design is provided that is able to discriminate multiple signalsby synchronizing with a modulated or pulsed stimulus light. The lightstimulus may be monochromatic or polychromatic and the pulses may besimultaneous or time division multiplexed. Inventions for systemsresponding to each type of stimulus are disclosed.

An imaging device is provided containing pixels that can discriminatebetween multiple signals by synchronization with the stimulus source,arranged in an array and containing circuitry to address, process, andsynchronize the device.

A pixel containing a photodiode that is sensitive to polychromatic lightis attached to multiple integrating nodes. These nodes can all beconnected simultaneously or can be individually selected during theframe time. The frame time is the period between pixel interrogationswhere the contents are recorded externally.

The photodiode may be connected to each integrating storage node viatransfer gates. Charge is accumulated in the photodiode until thetransfer gate is asserted. Charge flows to the storage node via chargerepulsion, thermal diffusion and node fringing fields.

If the signals from each reactant can be separated in time within aframe period, by switching rapidly to each storage node in the pixelcell signal separation can be gained. An example representation of apixel layout capable of collecting up to four simultaneous or sequentialsignals is shown in FIG. 23. At the end of the frame time, localdetermination of the most likely reactant can be made with amplitudecomparators and this in-pixel processing feature is disclosed herein.

A method in accordance with the present invention provides increasedaccuracy in detecting a longer duration signal pulse via synchronizedhigh speed sampling of the slower pulse within an imaging frame time. Byknowing the sampling onset and duration, local averaging of the noisebefore, during and after the sample can be measured. This is similar toexisting methods used with lock in amplifiers and algorithms likediscrete saturation transforms to increase the detectivity of low signalto noise pulses.

An example of this is shown in FIG. 24. In this figure, a signal of only0.6 electrons is embedded in an environment with one electron ofbackground signal. This signal is sampled 6 times per frame. The lefthalf of the frame contains a signal during an incorporation event andthe right half of the chart does not contain any tagged signal. Thetagged signal is not apparent in the data series.

The signal is only active when there is an emitting signal (such as anincorporating nucleotide) and a pulse of stimulus: light. By pulsing thestimulus during the frame and measuring the local standard deviation,during both the active and inactive stimulus events, the embedded signalcan be extracted from a very noisy environment with a signal to noiseratio of less than 1. This is shown in FIG. 25. In this figure, thelocal standard deviations during the active illumination periods arecombined (shown in the white trace) and compared against the inactiveillumination periods (shown in the aqua trace). Clear discrimination ofthe incorporating period and the time when no incorporation is occurringis shown.

Interleaved sampling can extend this discrimination to n-varieties ofsignals. The samples corresponding to the variety of emitter sensitiveto the stimulus during that frame will provide higher signal and theother stimuli will provide background samples. With this method, ifincorporation is occurring in one of four varieties as an example, 25%of the samples will contain signal data and the balance providebackground data Also disclosed are methods to utilize the phase shift ofa carrier sinusoid to extract signals in extremely noisy environments.This method essentially creates a homodyne with an inherent low passfilter to reduce noise.

After determination of the most likely reactant, the amplitude andspecies identification can be read out from each pixel in the array upto each frame period. A layout of an example smart pixel is shown inFIG. 23, for example, a pixel with multiple parallel processing elementsand additional conditioning and logical circuits to compare eachcomponent and perform data reduction.

Further aspects of the present invention discriminate species based uponfrequency differences is disclosed. In this case, each species can beidentified by an unique modulation frequency. The signal is recoveredwith a local mixer circuit embedded in each transfer node of the pixel.A compact low voltage circuit without lumped parameter inductancedesigned for CMOS device implementation is shown in FIG. 26 and is knowncommonly as the Gilbert mixer. This down converting mixer takes twoinputs, the photodiode output and a local oscillator signal broadcast toall pixels. The top transistor (M1) operates in the linear region whileM2 operates in saturation. The current flow through M1 and M2 istherefore equal.

In various embodiments, system designs may utilize multiple illuminantsthat rely on the separation of dye absorption regions for maximumdiscrimination. By separating the dye absorptions, each dye can be tunedto the stimulus wavelength with maximum efficiency. As an example. fourcommercial dyes are detailed in the table below. There are four stimuluswavelengths used and the relative responses for each wavelength aretabulated. In all cases, the contrast in absorption is nearly 4:1 orgreater. Using this approach, there is a one to one response from thedye to its corresponding stimulus with low contamination from otherdyes.

Stimulus Alexa488 Alexa532 Alexa610 Alexa750 488 nm 1 0.28 0.03 0 532 nm0.01 1 0.09 0.01 610 nm 0 0 1 0.09 750 nm 0 0 0 1

The absorption of each illuminant by these four dyes is shown in FIG.27. In this figure, the cross talk is shown by the reduced power peaksunderneath each curve for the matched dye. The 488 nm absorption ofAlexa488 is shown in the blue curve. Absorption of Alexa532 of nearly29% of the. energy is shown in red. The longer wavelength excitationshave considerably less absorption cross talk.

Vertical Detector

The current excitation/detection side of many current systems isgenerally very complex with numerous lenses, dichroics, holographicphase masks and other elements that are expensive, difficult to keep inproper alignment and increase the time-between-failures due to theirsheer number. Here we combine the waveguide illumination of the ZMW witha photonics band gap rejection layer and one novel wavelength-sensitive“pixel” per ZMW to measure emission. Registration issues between ZMW,optics and camera are removed by the introduction of this single rigidsolid state device.

Various aspects of the detectors may be improved in accordance with thepresent invention. With reference to FIG. 28, a reaction cell may beprovided in a cladding layer such as an Al/AlOx layer (1) with orwithout a micromirror. Micromirrors are described, for example in U.S.patent application Ser. No. 12/567,526 which is incorporated byreference herein in its entirety for all purposes. In the event thatwaveguide problems arise, such problems may be compensated for withmicrolens in photonic band gap (PBG) layer (3). A fused silica (fusi)waveguide (2) with Al metal layer on top and a PBG layer (3) below,potentially with high index PBG “bulge” into fusi to collimate emission(e.g., microlens or “Wens” (2.1)). A 1-D photonic band gap sandwich (3)of complex layers of low and high index material may be designed toblock pump lines and pass emission lines. Wavelength sensitivesingle-pixel detector with (e.g., (4)) or without grounded metal “miniFaraday” shielding (4.1) may be configured reduce ZMW-to-ZMW crosstalk.

In some cases, the waveguide (2) is a single mode fiber for deliveringillumination light. While the waveguide is shown spaced away from theZMW, in some cases, the waveguide can be adjacent to the ZMW. The photonband gap, or dielectric stack (3) can also be disposed within a singlemode core, and can be configured so as to reject stray illuminationlight to keep it from the detector below, acting as an in-line Braggfilter to screen out excitation light. Having a single mode fiber aspart of the detection optics is made possible because the fluorescentdipole in the ZMW is generally very small, and thus the emissions fromthe fluorophores occupy a very small space in phase volume. This resultsin a relatively high efficiency of coupling the emitted light into asingle mode waveguide.

In some cases there can be two different PBG layers or dielectricstacks, one in the path of the illumination light acting as aperpendicular coupler, and the other acting to pass emitted light, andreflect any stray illumination light back up into the ZMW, or back upinto the first PBG layer to couple back into the path of theillumination light.

Turning now to FIG. 29, a standard PIN junction (5) may have two or moreI-N junctions at different depths. A variation of this design can beproduced to have color-specific low-concentration dopants at varyingdepths to enhance regional absorption. Long wavelength photons thatpenetrate deeply into the I region, cause significant current from N2 inaddition to current from surface-proximal NI (e.g., (5.1)), whereasshort wavelength photons are absorbed quickly as a function of depth,and the primary measured current is from N1, with little from N2 (e.g.,(5.2)). This device allows for one detector or pixel to distinguishmultiple wavelengths, and therefore detect multiple differentfluorophores.

Other aspects of the present invention are directed to the provision ofthermoelectric (TE) pad arrays near the solution surface, or even in thealuminum metal that forms the ZMW, and other, cooling, pads below thedetector to reduce dark current (6) (see, e.g., FIG. 30). Although thismay cause significant thermal stresses in the composite chip, it may bedesired for implementation for at least two reasons: 1) thermalstability which is critical for proper enzymatic function, bufferstability, and corrosion so the TEs could serve to reduce the localtemperature due to, e.g., laser-induced heating; and 2) even a slighttemperature decrease for the detector region would benefit signal tonoise.

Spectral Dispersion

The following describes a family of spectral disambiguation solutionsrelevant to “integrated” optical platforms, in which free air optics arcminiaturized and condensed into a single monolithic device. Thesedevices and methods allow for obtaining information from multiplefluorophores from a single integrated analysis element.

With reference to FIG. 31, the fundamental concept is to convertemission profiles per channel from being isotropic (“Normal”) in tospatially unique (“Unique”) and project these onto a multi-pixel array.Put differently, these are techniques to create a unique pattern on thedetector depending on the color of light.

In some embodiments, emission from each channel is restricted using fourphotonic band gap stacks, as shown in FIG. 32, each only allowing onechannel through to a spatially-sensitive sensor (“quad photodiode” or“position sensitive diode”). Each element of the PBG stack will have thefeature not only that it will only allow light through of the color forwhich the detector is assigned, the elements will reflect back thewavelengths that are not passed through. The reflected photons canbounce back off of the bottom of the cladding layer and again encounterthe PBG stack. If the photon is not allowed through that element, it isreflected again. This feature provides for recycling of detectedphotons, which can be very useful in these types of systems where thenumber of photons to be measured can be low, and improvements in photoncapture efficiency are important. In some embodiments a can, reflectivetunnel, or cladding tunnel is provided around the PBG stack. The cancomprises a set of reflective walls around the PBG stack which receiveslight from a particular ZMW. The can will have a spherical, square, orany other suitable profile, and can have straight or curved walls. Thewalls can comprise any reflective material including metals ordielectric stacks. Because the walls are reflective, they are also partof the path of some of the light rays emitted from the ZMW. Thereflection of the walls of the can provide a further increase inefficiency of use of the photons reflected from the PBG stack asdescribed above.

Thus, in some aspects, the invention provides an integrated device formeasuring optical signals from an array of optical sources over time,the device comprising an array of elements, each element comprising: atop layer comprising an optical source that emits two or more opticalsignals, each optical signal comprising different wavelengths; a middlelayer comprising a spectral diversion element comprising a PBG stackhaving two or more photon band gap (PBG) elements, each PBG elementconfigured to allow light of a different transmission wavelength rangethrough the element and to reflect light that is not in the transmissionwavelength range; and a bottom layer comprising a detector; wherein eachPBG element transmits light onto a different detection region of thedetector, whereby the identity of the optical signal can be identifiedby the regions of the detector onto which light is transmitted.Generally the integrated device is configured such that some of thereflected light from one PBG element is reflected into another PBGelement through which it is transmitted, thereby increasing theefficiency of photon detection compared to where no reflected light isdetected.

The efficiency of reflection of light back into the PBG stack for photonrecycling can be improved by providing a can corresponding to eachoptical source, e.g. ZMW, comprising walls of reflective materialdisposed in the middle layer, the can configured to reflect some lightreflected by the PBG stack back to the PBG stack.

In other embodiments, optical path length (OPL) differences may createunique interference patterns for coherent emission from a single dye, asshown in FIG. 33. Two example paths have lengths ζ and ζ+δ from sourceto array. If that difference in distance is an integer multiple of thewavelength of light (λ), then constructive interference occurs and themeasured intensity (I) at that pixel is high; conversely, if δ˜nλ/2 thendestructive interference causes the measured intensity at that pixel tobe zero. In this manner, the measured pattern is related to the color oflight. FIG. 34 illustrates this effect for two colors. In some cases areflective can is created around each pixel in the region from thebottom of the cladding layer to the pixel array. The can comprises a setof reflective walls around the pixels which receive light from aparticular ZMW. The can have a spherical, square, or any other suitableprofile, and can have straight or curved walls. The walls can compriseany reflective material including metals or dielectric stacks. Becausethe walls are reflective, they are also part of the path of some of thelight rays emitted from the ZMW. Thus, the presence of these walls cancontribute to the constructive and destructive interference and thus canbe used to control the way that colored light can be directed todifferent pixels at different intensity.

In some cases, a reflective can is disposed around multiple pixelsdisposed over a plurality of ZMWs, where each of the pixels detectslight from more than one ZMW. Here, the destructive and constructiveinterference from the signals can be used to identify position as wellas identifying wavelength.

With reference to FIG. 35, a holographic medium may be inserted betweenthe source and array, which controls the relative phase of light andcreates highly non-linear interference patterns as a function of color.The pattern at the array is two dimensional, such that a full 2-D pixelarray is essential for collecting as much light as possible whileresolving the underlying pattern. However, at a minimum, a 1-D array“stripe” or a 2-D “pie-slice” might contain sufficient informationdistinguish multiple colors. Further, the pixel density must besufficient to distinguish colors that lack significant spectralseparation.

Stacked Junctions to Resolve Dye Spectra

Some aspects of the present invention are directed to methods to resolvethe color of an emitted photon from a dye with a vertically stackedphotodetector. The absorption depth in silicon is dependent onwavelength and can be excited to provide this spectral filtering.Silicon photodetectors have been used extensively in many applications.The photon is generally absorbed in a depleted region fanned by areverse biased diode that is exposed to incoming light. Thephotoelectron is stored for readout. An important aspect is to generatea depletion region deep enough to gather the absorbed photons within adiffusion length of the extent. The absorption depth is dependent on thewavelength of the incident photon. Longer wavelengths are absorbeddeeper in the material up to the point in the infrared where silicon istransparent.

By designing a photodetector with distinct regions of detector based ondepth, spectral separation can be realized. Although similar sensorshave been described, for example, in U.S. Pat. No. 6,911,712 whichdescribes a stacked diode that is fabricated in standard CMOS processes.Methods according to the present invention leverage the principles thatdetermination of four distinct colors from marker dyes is an easier taskthan traditional imaging color fidelity and a reduced set of diodes canbe exploited. For example, with two stacked depletion regions sharing acommon node, four spectral signatures can be determined based on theratio of these two measurements. It is apparent that the depthseparation can be used to resolve the color of the photon by designingthe detector depletion widths to fall between these mean absorptiondepths.

For example, and with reference to FIGS. 41( a)-(d), a sacrificial toplayer may be utilized to etch a basic detector into a mesa structure asshown. The processing for this detector may be performed in a modifiedCMOS process.

An intrinsic region (i) is grown on top of the p-type starting material(p). N-type regions (n) are placed at two (or more) different depths inthe starting material layer (p). One way to accomplish this is to growthe lower (n) layer during the intrinsic layer deposition and the upper(n) layer diffused on the finished layer. Another approach would be toimplant the lower (n) layer and diffuse the upper (n) layer on thefinished intrinsic coat. The entire structure can then be masked and theintrinsic area around the pixel etched to the p-type substrate (stop)layer, as shown in FIG. 41( b). A mesa structure results with an uppern-layer suspended above the substrate (p). The CMOS circuitry can bepatterned using relatively low temperature processes (e.g., below 1000°C.) to form the switching and amplification processing adjacent to themesa. Finally a p+ contact diffusion is provided to the commonphotodiode cathode. The n-regions and the p+ contact may be metallized(see, e.g., FIG. 41( c) and bussed to the external I/O pads (see, e.g.,FIG. 41( d).

Such a circuit may provide depth based photoelectron detection. Theintrinsic layer is a high impedance path and isolating the field linesfrom each n-type pickup may be tuned via doping. An alternative layoutis to use a graded n-type doping instead of an intrinsic layer. Suchincreasing resistance provides a field which can separate electronsbased on depth.

CMOS Pixel for Detection of Fluorescent Lifetimes

Some aspects of the invention relate to methods of distinguishingdifferent fluorophores by their unique decay lifetimes. These tend to berelatively high speed events, and the devices of the invention arehighly multiplexed. The determination of high speed events at highresolution can be hampered by the need to distribute high speed clocksacross a large array. However, as described herein, taking advantage ofhigh speed drift regions in CMOS pixel circuits, the signature of shortterm events such as fluorescent decay can be made to discriminatebetween different tag data without clock distribution issues.

The detection of molecules has been successfully performed usingfluoroscopic dye and nanoparticle tags attached to the reactantmolecules. In applications like DNA sequencing, the identification of aDNA nucleotide is important to determine the sequence of the polymer. Asshown in FIG. 36, different fluorophores can respond to a stimulus withdifferent turn on and decay constants. Detection schemes that canutilize decay lifetimes to identify the species are in wide applicationin several applications. However, the present methods for determiningthe lifetime of the decay are not readily transferable to highresolution and high speed sequencing architectures.

In accordance with the present invention, a CMOS pixel is configured todetermine different species of chemical tags by means of processing highspeed lifetime decay signatures. This pixel uses a high field to drivephotocharge down a drift region. This charge is frozen by inhibiting thefield and is stored in a set of analog storage devices (i.e., chargetransfer gates). These regions can be interrogated to determine therelative or absolute decay rates to determine the species ofillumination.

Also disclosed are novel methods of utilizing charge binning to improvethe SNR of fluorophore time signatures and variable time resolutioncustomized at the time of use for specific lifetime profiles to gainmaximum discrimination.

A solid state sensor uses photoconversion of photons to charge carriersto sense the optical signal. These charge carriers are generallyintegrated in a capacitance element within the pixel that can be formedby a depletion region in a reverse biased junction. These charges can becontrolled by electro-dynamic or magnetic forces that can be applied bythe supporting circuitry. In various embodiments, a charge transferdevice such as a CCD, the charge packets are serially transferred fromone location to another by applying various potentials to gates in theproximity to the charge packet.

There are two main mechanisms of charge mobility in imaging devices.Diffusion causes charged particles to move from areas of higherconcentration to lower concentration areas. These particles also driftwhen under the influence of electric or magnetic fields. The equationsdescribing the charge flow (current density) for each of these effectsare described by:

J _(diffusion) =qD(dn/dx)  Eq. (2)

J _(drift) =σE  Eq. (3)

In various embodiments, a device uses the drift current mechanism torapidly transport incident photocurrent through a narrow depletionregion during all or a portion of the exposure time. The appliedelectric field causes the charged particles to move with a velocity thatcan be used to determine the arrival time of the particle based upon thedistance traveled in a given time period. The distance traveled can bevaried by controlling the applied voltage. It is disclosed that theapplied voltage can be varied during the exposure time to extend theevent duration or apply additional resolution the a smaller portion ofthe entire event.

By determining both the drift and the diffusion components, systematicvariations in doping density, chip temperature and mobility arecancelled to improve the accuracy and uniformity of the arraymeasurements.

A typical electron drift velocity is determined by the temperature, thetotal doping and the electron's effective mass. At low electric fields,the majority of electron collisions are with acoustic phonons andimpurities. At high electric fields, the number of collisions is moretemperature dependent and the carriers interact more with nonpolaroptical phonons and the electron reaches velocity saturation. This valuehas been derived from measured data and numerical simulations and isdescribed by:

v=((2.4×10⁵)/(1+0.8e ^(T/600)))*m/s  Eq. (4)

For example, at 30° C., the saturation velocity is about 100,000 m/s. Ifa drift region is designed with a length of 50 microns, the electronswill traverse this distance in 500 picoseconds. If the region is dividedinto 10 equal lengths, the time resolution of pulses traveling along thedrift region is 50 picoseconds.

In addition to the drift channel, a means to capture the time signatureof this signal is disclosed. In one example, an adjacent set ofcapacitors is placed next to the drift channel and the signal istransferred laterally at the end of the exposure time. This is shown inFIG. 37. A typical operation of this pixel requires that synchronizationwith the stimulus signal is used as gate to start the drift. A transfergate between the photodetector and the depletion drift region is shownthat can be used for this gating. The voltage across the drift regioncan be varied to reduce the electron velocity. The resolution per equalspaced stage is therefore L/vd where L is the stage length and vd is thedrift velocity. The voltage across the drift region needs to be stoppedto freeze the waveform at a specific time after the stimulus to capturethe waveform (if a fluorophore is present).

Soon after the waveform is frozen, it is transferred to the storagenodes across the transfer gates. It is disclosed that many pulses can besummed in the storage nodes to increased sensitivity. For example, afluorophore used to determine the incorporation of a reactant into apolymer can be active for a much longer time than the pulse decayduration (i.e., 50 milliseconds). If the pulse decay response were 10nanoseconds and a stimulus with high repetition rate were used, up to 5million pulses could be applied to each incorporation event and storedin the drift region elements (up to the charge storage capacity).

In another embodiment, segmentation of the drift region can be performedwith MOS gates above the region. The elements can then be sequentiallyread out similar to standard CCD readout methods (i.e., to a floatingdiffusion transimpedance amplifier). This prevents the diffusive mixingof the pulse after it is frozen. This method does not allow for thesequential missing of multiple stimulus response to increasesensitivity.

It is disclosed that this method performs temporal filtering of thestimulus as the stimulus in inhibited during the decay time. Typically,the stimulus signal is transferred down the drift region ahead of thedecay signal and does not mix. This temporal filtering provides aneffective method of optical filtering of a sequence without the need foroptical filters. This method is compatible with standard IC fabricationflows, as is shown graphically in FIG. 38. It is disclosed that thetiming of the pixel can be set to allow for the stimulus signal tocompletely transfer to the drift region to maximize the resolution ofthe decay pulse.

The detection of this stimulus at the end of the drift region mayautomatically synchronize the detector elements-with the source- andgate the transfer of the signal to the storage nodes. This activefeedback provides a higher level of synchronization and will result inhigher fidelity data with less jitter.

Various methods of operating the device may continuously operate thedrift region until a high intensity pulse is detected at the end of thedrift region (signifying a stimulus pulse). The transfer of the decaysignal. (received just after the stimulus in the drift region) is thenperformed and the system reset for the next event. This disclosed methodeliminates the need for electrical communication and synchronization ofthe stimulus with each optode element and significantly increases thetemporal uniformity of each element while reducing the complexity of thesystem.

The drift detection elements can be combined in the charge domain tofurther increase the signal to noise ratio at the expense of thetemporal resolution.

The real time monitoring of these storage elements can be used to detectthe presence of a fluorophore onset and the end of the event to assistin kinetic analysis of the incorporation. The storage nodes can beactively monitored and the ratio of the signals used to make real timeanalysis of the tagged species while the event is occurring. Using thesemethods, the full incorporation signal is integrated in a single sampleto dramatically increase the sensitivity of the measurement and reducethe device readout bandwidth. The samples are processed to produce asingle output signal with the tag species identification. This methodprovide for the decoupling of output bandwidth with in-pixel temporalresolution.

By making the drift length much greater than the photodetector length,the maximum temporal sensitivity is gained as the charge transportacross the photodetector is minimized.

With increased time resolution and samples, multiple superimposed decaywaveforms can be captured and deconvolved. As shown in FIG. 39, acombination of fluorophore example decays or simultaneously input to thedrift optode. Each has a specific decay signature independent of theintensity. This combination can be made to study simultaneous events toincrease the data density or to use multiple fluorophore combinations toincrease the available number of tags per dye set. A system designedwith this invention can be made to resolve multiple simultaneous eventswith high accuracy within an optode that is called in the micron scale.An array of these drift optodes can be designed to provide a massivemultiplex of sequencing data without any optical filtering.

The foregoing descriptions of specific exemplary embodiments of thepresent invention have been presented for purposes of illustration anddescription. They are not intended to be exhaustive or to limit theinvention to the precise forms disclosed, and obviously manymodifications and variations are possible in light of the aboveteachings. The exemplary embodiments were chosen and described in orderto explain certain principles of the invention and their practicalapplication, to thereby enable others skilled in the art to make andutilize various exemplary embodiments of the present invention, as wellas various alternatives and modifications thereof. It is intended thatthe scope of the invention be defined by the Claims appended hereto andtheir equivalents.

1. An integrated device for measuring optical signals from an array ofoptical sources over time, the device comprising an array of elements,each element comprising: a top layer comprising an optical source thatemits two or more optical signals, each optical signal comprisingdifferent wavelengths; a middle layer comprising a spectral diversionelement; and a bottom layer comprising a detector sensitive to spatialdistributions of light; wherein each spectral diversion element divertseach of the two or more optical signals onto different regions of thebottom layer, whereby the identity of the optical signal can beidentified by the relative spatial light intensity across the detector.2. The integrated device of claim 1 wherein the optical source comprisesa zero mode waveguide and the optical signals are emitted fromfluorescent labels corresponding to a chemical or biochemical reactionoccurring within the zero mode waveguide.
 3. The integrated device ofclaim 2 wherein the chemical or biochemical reaction comprises nucleicacid synthesis.
 4. The integrated device of claim 1 wherein the spectraldiversion element comprises an optical grating or holographic element.5. The integrated device of claim 1 wherein the spectral diversionelement comprises a Bragg grating disposed at an angle to the normal tothe central ray of the emitted light.
 6. The integrated device of claim1 wherein the detector includes multiple proximal pixels and wherein thepixels are in a linear array.
 7. The integrated device of claim 1wherein the detector includes multiple proximal pixels and wherein thepixels are in a two dimensional array.
 8. The integrated device of claim1 wherein the four different optical signals are emitted from eachoptical source.
 9. The integrated device of claim 8 wherein the detectorhas four pixels, each corresponding to one of the four optical signalswhereby the spectral diversion element diverts each of the colors to adifferent element.
 10. The integrated device of claim 1 wherein thespectral diversion element comprises a lens.
 11. The integrated deviceof claim 10 wherein the lens is cylindrically symmetrical and divertsdifferent wavelengths of light at different angles from the center ofthe lens resulting in a circularly symmetric pattern on the detector foreach set of wavelengths.
 12. The integrated device of claim 11 whereinthe detector comprises one central pixel and one or more pixelscomprising a circular ring around the central pixel.
 13. A pixelcomprising a photodiode having at least a first and a second transfergate, wherein the pixel is configured such that the photodiode sendscharge to one transfer gate for a first period of time, then send chargeto a second transfer gate for a second period of time before theremaining charge from the pixel is unloaded.
 14. The pixel of claim 13wherein the pixel further comprises a third transfer gate and a fourthtransfer gate wherein after the second period of time, charge is sent tothe third transfer gate for a third period of time, then charge is sentto the fourth transfer gate for a fourth period of time before thecharge from the pixel is unloaded.
 15. A system comprising: anexcitation light source that emits a first wavelength range for a firstexcitation time, then emits a second wavelength range for a secondexcitation time, the excitation light source providing excitation lightto a sample: a sample having a first fluorophore that is excited by thefirst wavelength range, and a second fluorophore that is excited by thesecond wavelength range; and a pixel comprising a photodiode having atleast a first transfer gate and a second transfer gate, wherein thepixel is configured such that the photodiode sends charge to the firsttransfer gate for a first collection time, then send charge to thesecond transfer gate for a collection time before the charge from thepixel is unloaded; wherein the system is configured such that the firstexcitation time corresponds to the first collection time and the secondexcitation time correlates to the second collection time, whereby theemission from the first fluorophore can be distinguished from theemission from the second fluorophore.
 16. The system of claim 15 whereinthe light source further emits a third wavelength range for a thirdexcitation time, then emits a fourth wavelength range for a fourthexcitation time, wherein the sample further comprises a thirdfluorophore that is excited by the third wavelength range, and a fourthfluorophore that is excited by the fourth wavelength range, and thephotodiode further comprises a third transfer gate and a fourth transfergate, wherein the pixel is configured such that the photodiode sendscharge to the third transfer gate for a third collection time, then sendcharge to the fourth transfer gate fora collection time before thecharge from the pixel is unloaded, wherein the system is furtherconfigured such that the third excitation time corresponds to the thirdcollection time and the fourth excitation time correlates to the fourthcollection time, whereby the emission of each of the four fluorophorescan be distinguished.
 17. The system of claim 16 wherein the excitationlight comprises a first laser that emits the first wavelength range anda second laser that emits the second wavelength range, a third laserthat emits the third wavelength range, and a fourth laser that emits afourth wavelength range.
 18. A method comprising: illuminating a samplewith a first wavelength range for a first excitation time, thenilluminating the sample with a second wavelength range for a secondexcitation time; wherein the sample comprises a first fluorophore thatis excited by the first wavelength range, and a second fluorophore thatis excited by the second wavelength range; and directing emitted lightfrom the sample to a single pixel that measures light for a firstcollection time and then measures light for a second collection time,such that the pixel separately stores charge related to each of thecollection times whereby the charge related to each of the collectiontimes can be separately read out; wherein the first excitation timecorresponds to the first collection time and the second excitation timecorrelates to the second collection time, whereby the emission from thefirst fluorophore can be distinguished from the emission from the secondfluorophore. 19-21. (canceled)
 22. An integrated device for measuringoptical signals from an array of optical sources over time, the devicecomprising an array of elements, each element comprising: a top layercomprising an optical source that emits two or more optical signals,each optical signal having a different rate of signal decay; a middlelayer capable of transferring light from the top layer to the bottomlayer; and a bottom layer comprising a detector having a single pixel;wherein the pixel measures the characteristic absorption depth of eachof the two or more different species' signals, allowing the pixel todistinguish the identity of each of the optical signals. 23-27.(canceled)
 28. An optics collection and detection system comprising: areaction cell; an illumination light source providing illumination lightto the reaction cell at a first wavelength λ1; a detector for detectingexcitation light at a second wavelength λ2; and a photon band gap (PBG)layer disposed between the reaction cell and the detector, wherein thePBG layer rejects light at the first wavelength λ1 but allows light atthe second wavelength λ2 travel toward the detector.
 29. An opticscollection and detection system according to claim 28, furthercomprising: a plurality of detectors for detecting excitation light,wherein one of said detectors detects light at the second wavelength λ2,and another of said detectors detects light at a third wavelength λ3; aplurality of PBG, stacks, each disposed between the PBG layer and arespective detector, wherein one of said PBG stacks rejects light at thethird wavelength λ3 but allows light at the second wavelength λ2 totravel toward said one detector, and wherein another of said PBG stacksrejects light at the second wavelength) λ2 but allows light at the thirdwavelength λ3 to travel toward said other detector.
 30. An opticscollection and detection system according to claim 29, wherein thesystem includes four detectors, each formed by a sensor quadrant of aquad photodiode (QPD), and four PBG stacks, wherein each PBG stack isoptically aligned with a respective sensor quadrant. 31-34. (canceled)35. An integrated device for measuring optical signals from an array ofoptical sources over time, the device comprising an array of elements,each element comprising: a top layer comprising an optical source thatemits two or more optical signals, each optical signal comprisingdifferent wavelengths; a middle layer comprising a spectral diversionelement comprising a PBG stack having two or more photon band gap (PBG)elements, each PBG element configured to allow light of a differenttransmission wavelength range through the element and to reflect lightthat is not in the transmission wavelength range; and a bottom layercomprising a detector; wherein each PBG element transmits light onto adifferent detection region of the detector, whereby the identity of theoptical signal can be identified by the regions of the detector ontowhich light is transmitted. 36-37. (canceled)